| 摘要: |
| 【目的】对前期鉴定所获得的东方蜜蜂微孢子虫的nce-miR-11248进行表达和序列验证，预测、分析其靶基因，并检测nce-miR-11248及其靶基因在东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中的表达谱，为进一步开展nce-miR-11248调控东方蜜蜂微孢子虫侵染的功能及作用机制研究提供参考。【方法】利用Stem-loop RT-PCR和Sanger测序验证nce-miR-11248的真实性；利用相关生物信息学软件预测nce-miR-11248的靶基因，并分析其编码蛋白的分子特性；使用MEME和TBtools软件预测SGT1蛋白的保守基序和结构域，通过Mega 11.0软件进行氨基酸序列多重比对，采用邻接法构建系统进化树。采集东方蜜蜂微孢子虫孢子侵染1,2,4,6和8 d的意蜂工蜂中肠组织，采用RT-qPCR检测nce-miR-11248及其靶基因SGT1的表达谱。【结果】nce-miR-11248在东方蜜蜂微孢子虫孢子中真实存在，其序列长度为25 bp。nce-miR-11248的靶基因为SGT1。SGT1蛋白分子式为C765H1213N195O241S4，分子质量约为17.10 ku，脂溶系数为82.67，等电点为5.50，亲水系数为-0.709，不含典型的信号肽和跨膜结构域，可同时定位于细胞核、线粒体和过氧化物酶体。东方蜜蜂微孢子虫、家蚕微孢子虫、泛胞虫、肠脑炎微孢子虫和蚱蜢脑炎微孢子虫的SGT1中均含有4个保守基序(Motif 1、Motif 2、Motif 3和Motif 4)和1个结构域(SGT1超家族结构域)。东方蜜蜂微孢子虫、家蚕微孢子虫、肠脑炎微孢子虫和蚱蜢脑炎微孢子虫的SGT1聚为一支，且东方蜜蜂微孢子虫与家蚕微孢子虫的SGT1进化距离最近。相较于侵染东方蜜蜂微孢子虫后1 d，侵染后2,4,6和8 d nce-miR-11248的表达量均显著下调(P＜0.05)；侵染后2,4,6和8 d SGT1的表达量均下调，其中侵染后4,6和8 d的表达量与2 d的差异达显著水平(P＜0.05)。【结论】nce-miR-11248在东方蜜蜂微孢子虫孢子中真实存在；东方蜜蜂微孢子虫与家蚕微孢子虫的SGT1亲缘关系最近；随着侵染时间的延长，nce-miR-11248及其靶基因SGT1在东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中的表达量均呈持续下降；nce-miR-11248和SGT1是潜在的参与调控微孢子虫侵染过程的因子。 |
| 关键词: 东方蜜蜂微孢子虫 意大利蜜蜂 nce-miR-11248 SGT1基因 表达谱 |
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| 基金项目:国家自然科学基金面上项目(32172792);国家现代农业产业技术体系建设专项(CARS-44-KXJ7); 福建农林大学硕士生导师团队项目（郭睿）;福建农林大学动物科学学院(蜂学学院)科研扶持项目(郭睿);福建省大学生创新创业训练计划项目(202110389027) |
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| Study on bioinformatics and expression pattern of nce-miR-11248 and target gene SGT1 in Nosema ceranae |
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ZHANG Kaiyao,ZHAO Haodong,ZHANG Yiqiong,et al
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| Abstract: |
| 【Objective】In this study,previously identified nce-miR-11248 in Nosema ceranae was expressed and verified,its target gene was projected and analyzed,and their expression patterns during the Nosema ceranae infection to Apis mellifera ligustica workers were assessed,aiming to provide references for further exploration of the mechanism underlying N.ceranae infection regulated by nce-miR-11248.【Method】Stem-loop RT-PCR and Sanger sequencing were used to prove the authenticity of nce-miR-11248.Related bioinformatic software were employed to predict its target gene and analyze molecular characteristics of the encoded protein.MEME and TBtools were utilized to predict conserved motifs and structural domains in the encode protein.Multiple alignment of amino acid sequences was conducted by Mega 11.0 software,followed by construction of phylogenetic tree with the neighbor-joining method.The midgut tissues of A.m.ligustica workers at 1,2,4,6 and 8 d post N.ceranae infestation were collected.RT-qPCR was used to determine expression profiles of nce-miR-11248 and the target gene.【Result】It was proved that nce-miR-11248 was truly existed in N.ceranae spores,and the sequence length was 25 bp.The target gene of nce-miR-11248 was SGT1.The molecular formula of SGT1 was C765H1213N195O241S4 with molecular weight of approximately 17.10 ku,its lipolysis coefficient,isoelectric point and hydrophilic coefficient were 82.67,5.50 and -0.709,respectively.There was no classical signal peptide and transmembrane domain in SGT1,and it simultaneously located in nucleus,mitochondrion and peroxisome.Additionally,SGT1 proteins inN.ceranae,Nosema bombycis,Pancytospora,Encephalitozoon intestinalis and Encephalitozoon romaleae contained four conserved motifs of Motif 1,Motif 2,Motif 3 and Motif 4 and one structural domain of SGT1 superfamily.Furthermore,SGT1 proteins in N.ceranae,N.bombycis,E.intestinalis and E.romaleae were grouped into one clade,and N.ceranae SGT1 and N.bombycis SGT1 had the closest evolutionary distance.As compared to 1 day post N.ceranae inoculation,the expression level of nce-miR-11248 was significantly down-regulated at 2,4,6 and 8 days post inoculation (P<0.05),the expression level of SGT1 was down-regulated at 2,4,6 and 8 days post inoculation,and the differences in expression level at 4,6 and 8 days post inoculation reached significant levels (P<0.05).【Conclusion】This study showed that nce-miR-11248 was truly existed in N.ceranae spores.N.ceranae SGT1 and N.bombycis SGT1 had the closest genetic distance.Both nce-miR-11248 and target gene SGT1 presented continuous decreases during the N.ceranae infection to A.m.ligustica workers,and nce-miR-11248 and SGT1 were potential factors involved in modulation of the microsporidian infection process. |
| Key words: Nosema ceranae Apis mellifera ligustica nce-miR-11248 SGT1 gene expression pattern |
